QC Report


general
Report generated at2025-02-21 23:26:55
TitleAdrenalGland
DescriptionATAC-seq data from Liu et al. adrenal gland data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomemm10
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads98275020148372008
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads96799143146277744
Mapped Reads (QC-failed)00
% Mapped Reads98.598.6
Paired Reads98275020148372008
Paired Reads (QC-failed)00
Read14913751074186004
Read1 (QC-failed)00
Read24913751074186004
Read2 (QC-failed)00
Properly Paired Reads92921474140562738
Properly Paired Reads (QC-failed)00
% Properly Paired Reads94.694.69999999999999
With itself96591404145940148
With itself (QC-failed)00
Singletons207739337596
Singletons (QC-failed)00
% Singleton0.20.2
Diff. Chroms108991179857
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads3758493459140850
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads2829503745145531
Paired Optical Duplicate Reads00
% Duplicate Reads75.282976.3356

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads73310142120830578
Rm = Number of Mitochondrial Reads2875284331204872
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.2817166576109840.20524734198504363

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads1767198627095634
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads1767198627095634
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads1767198627095634
Paired Reads (QC-failed)00
Read1883599313547817
Read1 (QC-failed)00
Read2883599313547817
Read2 (QC-failed)00
Properly Paired Reads1767198627095634
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself1767198627095634
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.7278034452920570.7097313482990993
Fraction of reads in NFR (QC pass)TrueTrue
Fraction of reads in NFR (QC reason)OKOK
NFR / mono-nuc reads2.85705244577336932.6314509980747496
NFR / mono-nuc reads (QC pass)TrueTrue
NFR / mono-nuc reads (QC reason)OKOK
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.54049714616116150.5633351852922135
Fraction of Reads in blacklist regions0.0022776726962096960.0024203161291594063
Fraction of Reads in promoter regions0.157135593022764950.17270365402780388
Fraction of Reads in enhancer regions0.383014393515250630.39042286296013595

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments2934841650236624
Distinct Fragments885183613587085
Positions with Two Read18608012571892
NRF = Distinct/Total0.3016120.270462
PBC1 = OneRead/Distinct0.2483190.219265
PBC2 = OneRead/TwoRead1.1812551.158356

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt10022847792
N17211631978
N28813041224
Np9891648263
N optimal10022848263
N conservative10022847792
Optimal Setrep1_vs_rep2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.0132637793683531.0098552058921995
Self Consistency Ratio1.22205890509734321.2891362811933205
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks146608167556

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile171.0182.0508.0312.0
50 percentile (median)239.0268.0739.0481.0
75 percentile422.0490.01005.0771.0
Max size2023.02317.02676.02676.0
Mean347.6911219033068388.22946358232474782.3297142738744580.7241090314084

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment18.36511762880365618.997852749462098

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.158604125929452360.16536612401022868
Synthetic AUC0.48853033429799420.4907629729562188
X-intercept0.352984814976264660.2647561078028706
Synthetic X-intercept2.7403761601626088e-641.0483506307287832e-99
Elbow Point0.69351207163395980.7197789233310271
Synthetic Elbow Point0.50917552066513450.5078636334415639
Synthetic JS Distance0.38255712304543890.4211118149491841

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.26226293977371870.29807879749187640.292488768100114150.295682891518028960.292118191143676930.294662549299459060.28673034215354760.27452124776601950.2745352354702113

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.25292635614759060.21263077053139360.254114518966413570.25329079365845225

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.19758048339402450.156067688147783740.19449779252258870.19866293986591202

For macs2 raw peaks:


For overlap/IDR peaks: